Circadian Phase Modulates Embryonic Susceptibility to Bisphenol A-Induced ASD-Related Behavioral Alterations via nr1d1

Emerging evidence links environmental exposures and circadian dysregulation to autism spectrum disorder (ASD), yet whether circadian phase modulates vulnerability to developmental toxicants remains unclear. Here, we investigated whether embryonic bisphenol A (BPA) exposure induces circadian phase-dependent ASD-related behavioral alterations via disruption of nr1d1 rhythmicity in zebrafish. In control larvae, nr1d1 exhibited significant circadian oscillation, whereas BPA exposure reduced expression levels and dampened oscillation amplitude. Two-way ANOVA revealed significant treatment × phase interactions in nr1d1 expression. Pharmacological activation of Nr1d1 partially restored rhythmic expression. Behavioral assessments conducted at defined circadian phases demonstrated a significant treatment × phase interaction in social preference. BPA-exposed larvae exhibited reduced social preference selectively at circadian time 15 (CT15), corresponding to the trough phase of nr1d1 expression, whereas no differences were observed at circadian time 3 (CT3). In contrast, tactile hyper-responsiveness showed a significant treatment effect but no phase interaction. BPA exposure also induced phase-dependent alterations in ASD-related genes, including α-nrxn2a and β-nrxn3a, with significant treatment × phase interactions. At the molecular level, BPA increased reactive oxygen species, impaired antioxidant defense, enhanced neuroinflammatory responses, and disrupted excitatory–inhibitory balance. Several of these endpoints exhibited phase-dependent modulation and were partially attenuated by Nr1d1 activation. These findings indicate that circadian phase modulates embryonic susceptibility to BPA-induced ASD-related behavioral and molecular alterations. Disruption of nr1d1 rhythmicity may contribute to time-of-day-specific neurodevelopmental vulnerability following environmental exposure.